Substantiating the Modulatory Role of Reactive Oxygen Species in Stem Cell Specification: ROS Signaling Regulates Primordial Germ Cell Fate in Drosophila embryos in a Cell-Autonomous Manner

Abstract

Stem cells have two distinct fates that set them apart from all other cell types, the ability to self-renew and the ability to differentiate. The balanced specification of SCs in organ systems is denoted by appropriate amounts of self-renewing and differentiated cells, and is essential for proper growth and development of an organism, particularly the passing of the genome from one generation to the next via the germline SCs. One established regulator of SC specification is a toxic class of signaling molecules called reactive oxygen species, which are produced during oxidative metabolism. The specification of Drosophila primordial germ cells from an undifferentiated identity to a differentiated fate is an in vivo migratory process that is a model for analyzing factors that shape proper SC fate. PGC migration allows us to visually observe defects as PGCs move from their determined location in early development to ultimate coalescence with somatic gonadal precursor cells. Proper PGC specification and migration is necessary to form the embryonic gonad where PGCs differentiate into the germline stem cells which will form the haploid gametes of the fly. We obtained results that add credence to the literature that ROS play a prominent regulatory role in SC specification by characterizing the role ROS play in Drosophila PGCs. Our results demonstrate that ROS has a similar regulatory role in PGCs as they do in other analyzed SC systems. We found that the ROS accumulate in PGCs in a spatiotemporal manner, are always maintained above the baseline amount that appears early in development, and increase until PGCs reach their differentiated fate in the gonad. Genetic knockdown of the ROS scavenging protein SOD resulted in an increase in ROS generation as well as PGC number and migration defects in the Drosophila embryo, which suggests that ROS have a modulatory function in maintaining PGC proliferation and proper migration. Decreased levels of the Drosophila regulatory gene pxt resulted in defective PGC adhesion and migration as well. By overexpressing hopscotch, a component of the JAK/STAT pathway, which has an established role in SC fate decisions and PGC adhesion, we obtained similar phenotypic results that denote JAK/STAT may also be functionally connected to mechanisms underlying generation of ROS in PGCs. Taken together, we have determined that ROS are likely signaling to PGCs in a manner that downregulates cellular machinery that maintains the undifferentiated fate of PGCs, and are doing so in a cell-autonomous manner through some underlying connection with the JAK/STAT pathway and the regulatory gene pxt.